Separation and culture method of adipose-derived stem cells

A technique for adipose stem cells and culture methods, applied in cell dissociation methods, cell culture active agents, culture processes, etc., can solve the problems of increasing the cost of collagenase, achieve rapid proliferation, maintain cell stemness and biological characteristics, The effect of high separation efficiency

Active Publication Date: 2019-08-02
济南赛尔生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other methods such as tissue block attachment method, adsorption column method, direct centrifugation method and mechanical shaking method, this method has the advantage of high stem cell yield, but the use of collagenase also increases the cost

Method used

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  • Separation and culture method of adipose-derived stem cells
  • Separation and culture method of adipose-derived stem cells
  • Separation and culture method of adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Obtaining a new collagenase and its expression, purification and activity identification

[0039] 1. Obtaining collagenase

[0040] According to the collagenase gene sequence published by NCBI, a pair of primers (as shown in Seq ID No.7 and Seq ID No.8) were designed, and Bacillus cereus (Bacillus cereus) strain ACCC10263 (purchased from China Agricultural Microbiological Culture Collection Management Center) genomic DNA as a template for PCR amplification, electrophoresis and recovery of about 3.0kb band, the nucleotide sequence Seq ID No.1 of the collagenase encoding gene was obtained by sequencing, and the amino acid sequence of collagenase encoded by it was It is Seq ID No.2.

[0041] 2. Construction of engineering bacteria

[0042] The collagenase coding gene shown in Seq ID No.1 was connected to the multiple cloning site of the expression vector pET28a to obtain the recombinant expression vector Z1. The recombinant expression vector Z1 was transforme...

Embodiment 2

[0071] Embodiment 2, the acquisition of mutants and their expression, purification and activity identification

[0072] 1. Acquisition of Mutants

[0073] According to the functional region of collagenase, design some collagenase mutants that have 2-3 amino acid replacements in the specific position of amino acid sequence Seq ID No.2 with the collagenase obtained in Example 1, wherein four mutants (A, B, The mutation site information of C and D) is as follows:

[0074] Mutant A replaces amino acid d(Asp) with m(Met) at position 254 of the collagenase Seq ID No.2 sequence, and replaces amino acid r(Arg) with g(Gly) at position 255; the mutation The amino acid sequence of body A is shown in Seq ID No.3, and the nucleotide sequence of its encoding gene is shown in Seq ID No.4;

[0075] Mutant B replaces amino acid k (Lys) with l (Leu) at position 382 of the collagenase Seq ID No.2 sequence, and replaces amino acid e (Glu) with p (Pro) at position 383;

[0076] Mutant C replace...

Embodiment 3

[0089] Embodiment 3, the application of mutant A and D in the separation and cultivation of adipose stem cells as collagenase

[0090] 1. Influence of the amount (concentration) of mutants A and D on the separation effect of adipose stem cells

[0091] Using the mutants A and D prepared in Example 2 as collagenases, the adipose stem cells were separated according to the following steps, wherein the digestion time was the same, the amount (concentration) of collagenase added was different, and commercialized type I collagen Enzyme (invitrogen, Cat. No. 17100-017) as control (CK1):

[0092] (1) Negative pressure liposuction collects human adipose tissue, divides it into 100mL portions, sterilizes the surface of each container containing adipose tissue, and carefully transfers it to a biological safety cabinet. After standing still, the swelling fluid and adipose tissue Layer, and remove the lower liquid and upper layer of grease;

[0093] (2) Add an equal volume of normal saline...

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Abstract

The invention discloses a separation and culture method of adipose-derived stem cells. The method comprises the following steps of (1) collecting adipose tissues; (2) adding collagenase into the adipose tissues obtained in the step (1) for digestion to obtain the adipose-derived stem cells; (3) enriching the adipose-derived stem cells obtained in the step (2); and (4) conducting purification and subculture on the adipose-derived stem cells obtained in the step (3). In the step (2), an animo acid sequence of the collagenase is as shown in Seq ID No.5; a volume percent fraction of the adipose tissues in a digestion system is 40-60%; the content of the collagenase in the digestion system is 0.20-0.30 g / L; and digestion time is 15-30 min. The method has the advantages that the dosage of the collagenase is little, and the separation efficiency of the adipose-derived stem cells is high, so that the method has important values for scientific research and large-scale separation and culture ofadipose-derived stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing fat stem cells. Background technique [0002] Due to congenital deformity, trauma, tumor resection and other causes of soft tissue defects and the increasing demand for plastic surgery, soft tissue filling is a common technique used in plastic surgery. As an ideal soft tissue filling material, autologous fat is better in biocompatibility than artificial tissue substitutes, allogeneic and heterogeneous materials, without immune rejection, and has the advantages of easy material acquisition, simple operation, and good filling shape. However, the low postoperative fat survival rate (25%-80%) of autologous fat transplantation greatly limits its wide application in clinic. In recent years, with the gradual understanding of the survival mechanism after fat transplantation, people use adipose-derived stem cells to help transplanted fat have a higher su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00C12N2501/11C12N2501/165C12N2501/2303C12N2500/84
Inventor 宋珂慧胡振生刘振中庄秀萍郭伟
Owner 济南赛尔生物科技股份有限公司
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